p56Lck Kinase Inhibitor Screening Assay Kit

Intended Use

The p56Lck Kinase Inhibitor Screening Assay Kit is designed to measure the activities of recombinant catalytic domain of Lck for the rapid and sensitive evaluation of inhibitors or activators. The phosphotyrosine specific monoclonal antibody used in this assay kit has been demonstrated to recognize the phosphotyrosine residue in the recombinant "Tyrosine kinase-substrate-1", which is efficiently phosphorylated by p56Lck in vitro.

Applications of this kit include:
1) Screening inhibitors or activators of recombinant catalytic domain of Lck.
2) Detecting the effects of pharmacological agents on recombinant catalytic domain of Lck.

This assay kit is for research use only and not for use in diagnostic or therapeutic procedures.

Storage

Upon receipt store all components at 4°C.
Don't expose reagents to excessive light.

Performance Characteristics

The p56Lck Kinase Inhibitor Screening Assay Kit has been shown to detect the kinase activity of recombinant catalytic domain of p56Lck. The assay shows good linearity of sample response.

General Description

Lck is a 56-kDa protein tyrosine kinase that is predominantly expressed in T lymphocytes. A member of the Src kinase family, it has a unique N-terminal region followed by SH3, SH2, and catalytic domains. Lck is an important protein tyrosine kinase in lymphocytes; its overexpression renders T cells hypersensitive to antigen stimulation, and an Lck-deficient T cell line, J.CaM1, exhibits dramatically reduced protein tyrosine phosphorylation following T cell receptor (TCR) cross-linking. Furthermore, genetic experiments have shown that mice deficient in Lck or expressing a dominant-negative mutant form of Lck exhibit a severe defect in T cell maturation. Lck is localized to the membrane through myristylation and palmitylation and a portion of cellular Lck associates with the cytoplasmic tail of CD4 via cysteine residues. CD4 binds to class II major histocompatibility complex molecules on antigen-presenting cells, and this interaction between CD4 and major histocompatibility complex activates Lck, perhaps through conformational changes. The Lck associated with CD4 propagates key biochemical signals in CD4 co-receptor function. Like all Src family kinases, Lck is activated and inhibited by tyrosine phosphorylation, Tyr-394 is the site of stimulatory phosphorylation, whereas Tyr-505 is the site of inhibitory phosphorylation.

Measurement of Lck Kinase activity

The protocol generally regarded as most sensitive for the quantitative measurement of Lck kinase activity involves incubation of the Lck kinase sample with substrate, either a natural or synthetic polypeptide (such as poly[Glu,Tyr]4:1), in the presence of Mg2+, Mn2+ and 32P-labeled ATP. The reaction is terminated by "spotting" a sample onto a filter paper disc, followed by immersion in acid to precipitate the radiolabeled product. The filter papers are then washed extensively to remove unincorporated radiolabel and the radioactivity is counted. While sensitive, this method is labor-intensive, generates hazardous radioactive waste, and depends on a radioisotope of short half-life. It is particularly unsuitable when kinase assays are only performed on an infrequent basis. The Research Product p56Lck Kinase Inhibitor Screening Assay Kit uses a horseradish peroxidase coupled anti-phosphotyrosine monoclonal antibody as a reporter molecule in a 96-wells ELISA format. This assay provides a non-isotopic, sensitive and specific method to detect kinase activity of recombinant Lck catalytic domain.